U.S. patent number 4,235,961 [Application Number 06/050,518] was granted by the patent office on 1980-11-25 for method for photometric determination of the subunit b of creatine kinase and a reagent for carrying out the method.
This patent grant is currently assigned to LKB-Produkter AB. Invention is credited to Arne T. Lundin.
United States Patent |
4,235,961 |
Lundin |
November 25, 1980 |
Method for photometric determination of the subunit B of creatine
kinase and a reagent for carrying out the method
Abstract
A method for the photometric determination of the subunit B of
creatine kinase (CK-B) in buffered water solution through reaction
with creatine phosphate, ADP and the system luciferase/D-luciferin
in the presence of a specific antibody inhibiting the subunit M of
creatine kinase, comprises making the determination in the presence
of a D-luciferin analog, such as L-luciferin, having a structure
similar to D-luciferin and being a competitive inhibitor of the
firefly luciferase reaction. Also disclosed is a reagent for
carrying out the above method comprising creatine-phosphate, ADP,
luciferase, D-luciferin, L-luciferin, CK-M inhibiting antibodies,
diadenosinpentaphosphate, SH-reagent, magnesium salt and buffer as
well as possibly stabilizers and/or complex forming reagent.
Inventors: |
Lundin; Arne T. (Stockholm,
SE) |
Assignee: |
LKB-Produkter AB (Bromma,
SE)
|
Family
ID: |
6043140 |
Appl.
No.: |
06/050,518 |
Filed: |
June 21, 1979 |
Foreign Application Priority Data
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Jun 29, 1978 [DE] |
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2888658 |
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Current U.S.
Class: |
435/8; 435/184;
435/17; 435/810 |
Current CPC
Class: |
C12Q
1/50 (20130101); C12Q 1/66 (20130101); Y10S
435/81 (20130101) |
Current International
Class: |
C12Q
1/50 (20060101); C12Q 1/66 (20060101); C12Q
001/66 () |
Field of
Search: |
;435/8,17,184,810
;424/2 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
"Determination of Creatine Kinase Isoenzyme MB Activity in Serum
Using Immunological Inhibition of Creative Kinase", Subunt
Activity, Neumier Climica Chimica Acta 73 (1976) 445-451. .
"Immobilized Firefly Luciferase", Lee, Analytical BioChemistry 80,
(1977), pp. 490-501..
|
Primary Examiner: Therkorn; Ernest G.
Attorney, Agent or Firm: Fisher, Christen & Sabol
Claims
I claim:
1. Method for photometric determination of the subunit B of
creatine kinase (CK-B) in buffered water solution through reaction
with creatine phosphate, ADP and the system luciferase/D-luciferin
in the presence of a specific antibody inhibiting the subunit M of
creatine kinase, characterized in that the determination is made in
the presence of a D-luciferin analog having a structure similar to
D-luciferin and being a competitive inhibitor of the firefly
luciferase reaction.
2. Method according to claim 1, characterized in that
diadenosinpentaphosphate is added.
3. Method according to claims 1 or 2, characterized in that ADP in
a quantity of 10.sup.-5 to 5.times.10.sup.-3 M is added.
4. Method according to claim 1, characterized in that the method is
carried out at a pH value between 6.5 and 7.8.
5. Method according to claim 4, characterized in that a buffer
concentration between 10 mM and 250 mM is used.
6. Method according to claim 1, characterized in that a thiol
protecting reagent, preferably N-acetylcystein, glutathion,
dithiotreitol or dithioerythrit, is added.
7. Reagent for carrying out a method for the photometric
determination of the subunit B of creatine Kinase (CK-B),
comprising creatine-phosphate, ADP, luciferase, D-luciferin,
L-luciferin, CK-M inhibiting antibodies, diadenosinpentaphosphate,
SH-reagent, magnesium salt and buffer.
8. Reagent according to claim 7, characterized in that it
comprises:
9. Reagent according to claim 8, characterized in that it
comprises:
Description
The present invention refers to a method for photometric
determination of the subunit B of creatine kinase (CK-B) especially
in serum, and a reagent suitable for carrying out the method.
The creatine kinase enzyme (EC 2.7.3.2) catalyses the
transformation
In human tissue there exist two different subunits of this enzyme,
namely M and B. The active enzymatic unit always consists of two
subunits which together could form three different isoenzymes,
namely the muscular type (CK-MM), the brain type (CK-BB) and the
hybrid type (CK-MB) which in practice only appears in myocardial
tissue. However, at a heart infarction the hybrid enzyme CK-MB
appears also in serum. This isoenzyme CK-MB is therefore of great
importance in the diagnosis of acute myocardial infarction.
From Clinica Chimica Acta 73 (1976), pp 445 to 451 there is known a
method for determining the isoenzyme CK-MB, which eventually is
based on determination of the subunit CK-B. This is possible as the
brain enzyme CK-BB which always contains the subunit CK-B does
normally not appear in serum and thus consequently the CK-B
activity found in serum is derived from the isoenzyme CK-MB. In the
method known per se the determination takes place by determining
the ATP obtained in accordance with the above cited reaction in
accordance with the following equation: ##STR1## The determination
is specific for CK-MB as it takes place in the presence of
antibodies which specifically inhibit the activity of the subunit M
of the creatine kinase(CK-M).
An essential disadvantage of the method known per se is firstly a
too low sensitivity and secondly, the method includes a delay phase
which is dependant on temperature and which is not possible to
reduce to less than 90 seconds. This delay phase has the
consequence that when the method is used in automatic analysers
their capacity cannot be fully used and in certain fast analysers
the method cannot be used at all. Furthermore, the concentrations
of CK-B in serum at a heart infarction is very low and because of
the low sensitivity of the method known per se the activity
appearing cannot be determined with the required accuracy.
It is an object of the present invention to provide a method for
determining the subunit B in creatine kinase in which the above
mentioned disadvantages are eliminated. Thus, the method according
to the invention does not suffer from the lag phase and the
sensitivity is considerably increased in comparison with the method
known per se and furthermore, as the reaction is linear it makes a
kinetic measuring possible.
According to the invention the above object is achieved by means of
a method for photometric determination of the subunit B of creatine
kinase CK-B in buffered water solution by means of reaction with
creatine phosphate, ADP and the system luciferase/D-luciferin in
the presence of a specific antibody inhibiting the subunit M of the
creatine kinase, the method being characterized by that the
determination is carried out in the presence of a D-luciferin
analog, such as L-luciferin, having a structure similar to
D-luciferin and being a competitive inhibitor of the firefly
luciferase reaction.
A method for determining creatine kinase (not subunit B) by using
luciferase-D-luciferin-systems is known per se. (Analytical
Biochemistry 80, (1977) pp 496-501). This method uses purified
luciferase together with D-luciferin, ADP, creatine-phosphate,
magnesium sulphate and buffer. The light emission will however
thereby appear as a peak which complicates the practical use of the
method and makes kinetic determinations impossible. Only through
the combination of the method and the antibody inhibiting the
subunit M and the addition of L-luciferin it is possible to
determine the subunit B and simultaneously obtain a continuous
light emission proportional to the enzyme activity of the subunit B
which makes a kinetic monitoring of the light emission possible. It
was not foreseeable that in the presence of M subunit inhibiting
antibodies the luciferase reaction would be quantitative, nor was
it foreseeable that it was possible to transform the peak light
emission to continuous emission extending for several minutes and
thereby simultaneously eliminate the lag phase.
Luciferase is obtained from the American firefly (Photinus pyralis)
and is possible to purify by methods known per se. According to the
invention the luciferase substance of the highest possible purity
is used as unpurified luciferase substances contain interfering
substances. Luciferase catalyses the reaction
The amount of light emitted is directly proportional to the
ATP-concentration.
In the method according to the invention luciferase is suitably
used in concentrations between 0.5 and 20 .mu.g/ml, preferably in
quantities between 1 and 3 .mu.g/ml, these amounts referring to the
pure luciferase. As to the D-luciferin the amount in the test
solution is suitably between 25 and 500 .mu.g/ml, preferably
between 50 and 150 .mu.g/ml.
In accordance with the preferred embodiment of the invention
diadenosinpentaphosphate is added in amounts in the order of
5.times.10.sup.-8 to 5.times.10.sup.-6 M. Any remaining residual
disturbances from the myokinase are negligible particularly at low
concentrations of ADP. The ADP concentration is usually between
1.times.10.sup.-5 and 5.times.10.sup.-3 M, preferably between
1.times.10.sup.-5 and 5.times.10.sup.-4 M.
L-luciferin is preferably added in such an amount that at least a
25% inhibition of the luciferase reaction is obtained. The
inhibition should however not be above 95%. Preferably the amount
added is such that an inhibition between 50 and 90% is obtained.
The above suggested preferred ranges for the addition of luciferase
and D-luciferin gives an addition of L-luciferin suitably in the
order of 1 to 10 .mu.g/ml.
Additionally, a suitable magnesium salt, such as magnesium acetate,
is added. An addition of further stabilizing means for enzymes,
such as bovine serum albumin and/or complex generating reagents,
such as EDTA, is possible.
The type of buffer used is not critical. Suitable buffers are for
instance imidazol-acetate-buffer, Tris-acetate-buffer,
triethanolamin-acetate-buffer or phosphate-buffer. Preferably,
Imidazol-acetate-buffer is used. The pH determined by the buffer
should be in the order of 6.5 to 7.8, preferably from 6.7 to 7.0.
The buffer concentration could be varied within wide ranges. Thus,
a concentration from 10 to 250 mM could be used. Preferably 60 to
120 mM is used.
Suitably a thiol protecting reagent is added to the reaction
solution, such as N-acetyl cystein (NAC), Gluthathion,
Ditriotreitol or dithioerythrit. Preferably NAC is used. The
preferred concentration range for this reagent is between 2 and 50
mM.
A creatine phosphate is added in excess. A preferable range is 5 to
50 mM, preferably 8 to 15 mM.
A further object of the invention is to provide a reagent for
determining the subunit B of creatine kinase, comprising creatine
phosphate, ADP, luciferase, D-luciferin, L-luciferin, CK-M specific
antibodies, diadenosinpentaphosphate, SH-reagent, magnesium salt
and buffer as well as stablisers and/or complex forming
reagents.
The complex forming reagent to be used according to the invention
is for instance ethylendiamintetra acetic acid (EDTA) and
Nitrilotriacetic acid.
Suitably the reagent comprises in a lyophilisated or with water
recomposed form, the above defined components in the following
quantities.
TABLE
__________________________________________________________________________
preferred quantity suitable quantity
__________________________________________________________________________
Creatine-phosphate 8-15 mM 5-50 mM ADP 1 .times. 10.sup.-5 -5
.times. 10.sup.-4 M 1 .times. 10.sup.-5 -2 .times. 10.sup.-3 M
Luciferase 1-3 .mu.g/ml 0.5-20 .mu.g/ml D-luciferin 50-150 .mu.g/ml
25-500.mu.g/ml L-luciferin 1-10 .mu.g/ml 0.5-20 .mu.g/ml
CK-M-Antikorper.sup.(+) Diadenosinpentaphosphate 1 .times.
10.sup.-7 -1 .times. 10.sup.-6 M 5 .times. 10.sup.-8 -5 .times.
10.sup.-6 M Thiolreagent 2-50 mM 1-100 mM Magnesiumacetate 5-20 mM
1-100 mM Imidazol acetate buffer 60-120 mM 10-250 mM and possibly
Bovine serum albumin 0.5-2 g/l 0.1-10 g/l EDTA 0.5-10 mM 1-5 mM
__________________________________________________________________________
.sup.(+) sufficient to inhibit completely a CKMM-activity of 1000
U/1 sample solution at 25.degree. C.
The reagent can be supplied containing all its constituents or can
be supplied as a test kit as several separate solutions containing
different constituents. If the mixture contains all the
constituents, the reaction starts by adding the sample solution to
be determined, preferably a serum sample. If a preincubation with a
serum sample is required at least one of the constituents necessary
for the reaction for instance ADP and/or creatine phosphate are
added only at the end of the incubation period.
By means of the method and reagent according to the invention it is
possible to carry out the determination of the subunit B of
creatine kinase with considerably higher sensitivity as compared to
the method known per se. As the lag phase is eliminated the method
is more suited for automatic analysis equipment. The determination
of the subunit M would easily be carried out in the same way,
however without adding the antibodies specifically inhibiting the
subunit M whereafter the total activity of the creatine kinase is
determined and the activity of the subunit B determined according
to the invention is subtracted from the total activity.
Antibodies specifically inhibiting subunit M of creatine kinase are
known per se. Such antibodies are obtained through immunisation of
test animals suitably goats with the isoenzyme CK-MM and extracting
the antibody containing fraction from the serum. A method for
extracting these antibodies is described for instance in Clinica
Chimica Acta 73, (1976) p 446.
The following examples explain the invention in detail.
EXAMPLE 1
______________________________________ Concentration in End
concentration Solutions the solution in the test
______________________________________ (a) Luciferase reagent pure
luciferase 10 .mu.g/ml 2 .mu.g/ml D-luciferin 250 .mu.g/ml 50
.mu.g/ml L-luciferin 20 .mu.g/ml 4 .mu.g/ml Bovine serum albumin
(BSA) 5 mg/ml 1 mg/ml Magnesium acetate 50 mM 10 mM (b) Buffer
Imidazolacetate 143 mM 100 mM EDTA 2.9 mM 2 mM N-acetyl cystein
(NAC) 14.3 mM 10 mM (c) ADP-reagent ADP 5 .times. 10.sup.-4 M
10.sup.-5 M Ap.sub.5 A 5 .times. 10.sup.-6 M 10.sup.-7 M
Imidazolacetate 100 mM 100 mM (d) Creatine phosphate 0.5 M 10 mM
(e) ATP-standard-solution 10.sup.-5 M 2 .times. 10.sup.-7 m
______________________________________
Test Instructions
Constituants mixed:
0.2 ml luciferase reagent
0.7 ml buffer
10 to 50 .mu.l serum-sample
20 .mu.l CK-M-antibody solution
After 15 minutes incubation at 25.degree. C. is added:
20 .mu.l ADP-reagent and
20 .mu.l creatine-phosphate-solution.
The increase of the light emission is monitored by a commercially
available monitor and a printer. The rise is determined. Thereafter
a 20 .mu.l ATP-solution is added as an internal standard. The
obtained rise of the signal is used as a calibration.
FIG. 1 of the attached drawing shows the typical development with
respect to time of the light signal obtained.
EXAMPLE 2
To prepare a reagent according to the invention 2 mg luciferase, 5
mg D-luciferin, 400 .mu.g L-luciferin, 100 mg bovine serum albumin
1 m mol magnesium acetate, 10 m mol Imidazol-acetate, pH 6.7, 0.2 m
mol EDTA, 1 m mol N-acetylcystein, 10.sup.-6 mol ADP, 10.sup.-8 mol
diadenosinpentaphosphate and 1 m mol creatine phosphate are solved
in 94 ml water and lyophilisised.
To reconstituate the reagent before use is solved again in 94 ml
water. The amount of reagent is sufficient for carrying out 100
tests. The reagent is initially determined by addition of the
creatine kinase comprising serum sample.
EXAMPLE 3
Testkit: for 100 tests
I. Luciferase reagent
Lyophilisate is solved in 20 ml water and comprises:
2 mg luciferase
5 mg D-luciferin
400 .mu.g L-luciferin
100 mg bovine serum albumin
1 m mol Magnesium acetate
II. Buffer
Lyophilisate is solved in 70 ml water and comprises:
10 m mol Imidazol acetate, pH 6.7
0.2 m mol EDTA
1 m mol N-acetyl cystein
2 ml CK-M-antibody solution
III. ADP-reagent
Lyophilisate dissolved in 2 ml water comprises:
10.sup.-6 mol ADP
10.sup.-8 mol Ap.sub.5 A
200 m mol Imidazol acetate, pH 6.7
IV. Creatine phosphate
Lyophilisate dissolved in 2 ml water comprises:
1 m mol creatine phosphate
This test-kit enables a preincubation to be carried out with the
sample to be determined. The determination then follows example 1
above adding the corresponding amounts of the solvents III and
IV.
* * * * *